The transcriptional response of Arcobacter butzleri to cold shock.

Arcobacter (A.) butzleri is an emerging zoonotic pathogen associated with gastrointestinal diseases, such as abdominal cramps and diarrhea, and is widely detected in animals, showing a high prevalence in poultry and seafood. The survival and adaptation of A. butzleri to cold temperatures remains poorly studied, although it might be of interest for food safety considerations. To address this, growth patterns of eight A. butzleri isolates were determined at 8 °C for 28 days. A. butzleri isolates showed strain-dependent behavior: six isolates were unculturable after day 18, one exhibited declining but detectable cell counts until day 28 and one grew to the stationary phase level. Out of 13 A. butzleri cold shock-related genes homologous to Escherichia coli, 10 were up-regulated in response to a temperature downshift to 8 °C, as demonstrated by reverse transcription-quantitative PCR. Additionally, we compared these data with the cold-shock response in E. coli. Overall, we provide a deeper insight into the environmental adaptation capacities of A. butzleri, which we find shares similarities with the E. coli cold-shock response.

A critical rebuttal of the proposed division of the genus Arcobacter into six genera using comparative genomic, phylogenetic, and phenotypic criteria.

The proposal to restructure the genus Arcobacter into six distinct genera was critically examined using: comparative analyses of up to 80 Epsilonproteobacterial genome sequences (including 26 arcobacters); phylogenetic analyses of three housekeeping genes and also 342 core genes; and phenotypic criteria. Genome sequences were analysed with tools to calculate Percentage of Conserved Proteins, Average Amino-acid Identity, BLAST-based Average Nucleotide Identity, in silico DNA-DNA hybridisation values, genome-wide Average Nucleotide Identity, Alignment Fractions and G+C percentages. Genome analyses revealed the genus Arcobacter sensu lato to be relatively homogenous, and phylogenetic analyses clearly distinguished the group from other Epsilonproteobacteria. Genomic distinction of the genera proposed by Pérez-Cataluña et al. [2018] was not supported by any of the measures used and a subsequent risk of strain misidentification clearly identified. Similarly, phenotypic analyses supported the delineation of Arcobacter sensu lato but did not justify the position of the proposed novel genera. The present polyphasic taxonomic study strongly supports the continuance of the classification of "aerotolerant campylobacters" as Arcobacter and refutes the proposed genus-level subdivision of Pérez-Cataluña et al. [2018].

Arcobacter butzleri, un agente zoonótico en leche bovina / Arcobacter butzleri, a zoonotic agent in bovine milk / Arcobacter butzleri, um agente zoonótico em leite Bovino

Para la búsqueda de especies de Arcobacter fueron estudiadas 50 muestras de leche bovina obtenidas de los centros de acopio de la empresa láctea Ecolac, de las provincias de Loja y Zamora Chinchipe, Ecuador, y se aisló un total de ocho cepas de Arcobacter butzleri (16%). Esta frecuencia de aislamiento concuerda y está dentro de los rangos descriptos en la literatura. Todas las cepas aisladas fueron sensibles a gentamicina. Se encontró alta frecuencia de resistencia a tetraciclina (6/8 cepas) y a ciprofloxacina (4/8 cepas). Se verificó la ocurrencia de multirresistencia en tres de las ocho cepas aisladas.

A total of 50 samples of bovine milk obtained from bulk tanks milk of the collection centers belonging to the company ECOLAC, of the provinces of Loja and Zamora Chinchipe, Ecuador, were studied for Arcobacter species diagnosis, being isolated 8 strains of Arcobacter butzleri (16%). This frequency of isolation agrees and falls within the ranges described in the literature. All the isolated strains were susceptible to gentamicin. High resistance levels to tetracycline and ciprofloxacin were found with 6/8 and 4/8 resistant strains respectively. Multi-resistance was found in three of the eight isolated strains.

Foram estudadas, para a pesquisa de espécies de Arcobacter, 50 amostras de leite bovino, obtidas dos centros de coleta da empresa de laticínios ECOLAC, das províncias de Loja e Zamora Chinchipe, Equador, sendo isoladas em total 8 cepas de Arcobacter butzleri (16%). Esta frequência de isolamento concorda e está dentro dos níveis descritos na literatura. Todas as cepas isoladas foram sensíveis à gentamicina. Foi encontrada alta frequência de resistência à tetraciclina (6/8 cepas) e à ciprofloxacina (4/8 cepas), sendo verificada a ocorrência de multirresistência em três das oito cepas isoladas.

Arcobacter peruensis sp. nov., a Chemolithoheterotroph Isolated from Sulfide- and Organic-Rich Coastal Waters off Peru.

Members of the epsilonproteobacterial genus Arcobacter have been identified to be potentially important sulfide oxidizers in marine coastal, seep, and stratified basin environments. In the highly productive upwelling waters off the coast of Peru, Arcobacter cells comprised 3 to 25% of the total microbial community at a near-shore station where sulfide concentrations exceeded 20 µM in bottom waters. From the chemocline where the Arcobacter population exceeded 106 cells ml-1 and where high rates of denitrification (up to 6.5 ± 0.4 µM N day-1) and dark carbon fixation (2.8 ± 0.2 µM C day-1) were measured, we isolated a previously uncultivated Arcobacter species, Arcobacter peruensis sp. nov. (BCCM LMG-31510). Genomic analysis showed that A. peruensis possesses genes encoding sulfide oxidation and denitrification pathways but lacks the ability to fix CO2 via autotrophic carbon fixation pathways. Genes encoding transporters for organic carbon compounds, however, were present in the A. peruensis genome. Physiological experiments demonstrated that A. peruensis grew best on a mix of sulfide, nitrate, and acetate. Isotope labeling experiments further verified that A. peruensis completely reduced nitrate to N2 and assimilated acetate but did not fix CO2, thus coupling heterotrophic growth to sulfide oxidation and denitrification. Single-cell nanoscale secondary ion mass spectrometry analysis of samples taken from shipboard isotope labeling experiments also confirmed that the Arcobacter population in situ did not substantially fix CO2 The efficient growth yield associated with the chemolithoheterotrophic metabolism of A. peruensis may allow this Arcobacter species to rapidly bloom in eutrophic and sulfide-rich waters off the coast of Peru.IMPORTANCE Our multidisciplinary approach provides new insights into the ecophysiology of a newly isolated environmental Arcobacter species, as well as the physiological flexibility within the Arcobacter genus and sulfide-oxidizing, denitrifying microbial communities within oceanic oxygen minimum zones (OMZs). The chemolithoheterotrophic species Arcobacter peruensis may play a substantial role in the diverse consortium of bacteria that is capable of coupling denitrification and fixed nitrogen loss to sulfide oxidation in eutrophic, sulfidic coastal waters. With increasing anthropogenic pressures on coastal regions, e.g., eutrophication and deoxygenation (D. Breitburg, L. A. Levin, A. Oschlies, M. Grégoire, et al., Science 359:eaam7240, 2018, https://doi.org/10.1126/science.aam7240), niches where sulfide-oxidizing, denitrifying heterotrophs such as A. peruensis thrive are likely to expand.

Do the Escherichia coli European Union shellfish safety standards predict the presence of Arcobacter spp., a potential zoonotic pathogen?

The genus Arcobacter comprises Campylobacter-related species, considered zoonotic emergent pathogens, the presence of which in water has been associated with fecal pollution. Discharges of fecal polluted water into the sea have been considered as one of the main reasons for the presence of Arcobacter in shellfish, and this may represent a risk for public health. In this study, the European Union shellfish food safety criteria based on levels of Escherichia coli were studied in relation to their capacity to predict the presence of Arcobacter species. In addition, the accumulation factor (AF) that measures the concentration ratio between the microbes present in the shellfish and in the water, was also studied for both bacteria. The results show that the presence of E. coli correlated with the presence of the potentially pathogenic species A. butzleri and A. cryaerophilus. However, in 26.1% of the shellfish samples (corresponding to those taken during summer months) E. coli failed to predict the presence of, for instance A. butzleri and A. skirrowii, among other species. In the rest of the samples a significant correlation between the concentration of E. coli and Arcobacter spp. (mussels and oyster; R2=0.744) was found. This study indicates that the presence of E. coli can predict the presence of pathogenic Arcobacter species in shellfish samples harvested from water with temperatures lower than 26.2°C. Consumption of shellfish collected at higher temperatures which may not be permissive to the growth of E. coli but does allow growth of Arcobacter spp., may represent a risk for consumers.

Isolation and identification of Arcobacter species from environmental and drinking water samples.

Water plays an important role in the transmission of Arcobacter spp. to animals and humans. The aim of this study was to isolate and characterize Arcobacter spp. from 115 different water samples (66 sewage, 25 rivers, 16 spring water, and 8 drinking water) in Izmir, Turkey. In total, 41 samples (35.7 %) were found positive for Arcobacter spp. by the genus-specific PCR. Arcobacter butzleri was detected in 39 out of 115 samples (33.9 %) including 24 sewage, 13 rivers, and 2 spring water. The remaining Arcobacter spp. (n = 2) isolates could not be identified by m-PCR and 16S rRNA gene sequencing. Based on the phenotypic characterization, most of the Arcobacter species (87.8 %) indicated weak catalase activity. In addition, there were differences in phenotypic patterns among isolated species during growth at 37 °C under microaerobic and aerobic conditions, in the presence of 2 % (39/41) and 3.5 % (32/41) NaCl and 0.04 % TTC (39/41) and on MacConkey agar (38/41). The results of this study indicated that environmental water samples are common sources for Arcobacter spp. Therefore, effective control measures should be taken to protect human health.

Occurrence of potentially pathogenic arcobacters in shellfish.

Considering that several recent cases of human gastroenteritis have been associated with species from the Arcobacter genus, and that few data are currently available about the occurrence of this genus in Italian shellfish, the aim of the present study was to evaluate the occurrence of Arcobacter spp. and the presence of virulence-associated genes. The approach consisted of cultural and biomolecular (multiplex-PCR and 16S-RFLP) methods identifying isolates, followed by PCR assays aimed at the cadF, ciaB, cjl349, irgA, hecA putative virulence genes. Arcobacter spp. was detected in 16/70 (22.8%) shellfish samples. Specifically, Arcobacter spp. was highlighted in 10/42 (23.8%) mussel and in 6/28 (21.4%) clam samples. Subsequently, biomolecular assays revealed Arcobacter butzleri in 12/16 (75%) and Arcobacter cryaerophilus 1B in 4/16 (25%) isolates. PCRs aimed at the five putative virulence genes demonstrated widespread distribution of these genes among Arcobacter isolates and some differences from the results published by other authors. Our research provides more information regarding the health risks associated with the consumption of raw bivalve molluscs and underlines the need to implement an adequate control plan by performing intensive and continuous monitoring in order to guarantee human health.

SURVIVAL CAPACITY OF Arcobacter butzleri INOCULATED IN POULTRY MEAT AT TWO DIFFERENT REFRIGERATION TEMPERATURES.

Arcobacter spp. are emerging enteropathogens and potential zoonotic agents that can be transmitted by food and water, being considered a public health risk. The high isolation rate of these bacteria from poultry products suggests that it may be a major source of human infections. One hallmark for differentiating the genus Arcobacter from Campylobacter includes their growing capacity at low temperatures (15-30 °C) under aerobic conditions. However, little is known about the population density variation of these bacteria at different refrigeration temperatures. The aim of this study was to determine the survival behavior of two different Arcobacter butzleri concentrations (10(4) CFU/mL and 10(7) CFU/mL) inoculated on chicken legs and held at two different refrigeration temperatures (4 and 10 °C) throughout storage time. Results have shown that A. butzleri had growing capacity both at 4 and 10 °C. No statistical difference between the survival trends was found for both bacterial concentrations and temperatures tested. This study shows that A. butzleri is a robust species with regard to storage temperature, and represents a potential health risk for poultry meat consumers.

Occurrence and antimicrobial resistance of emergent Arcobacter spp. isolated from cattle and sheep in Iran.

This study is conducted to determine the occurrence and antimicrobial resistance of Arcobacter spp. isolated from clinically healthy food animals. A total of 308 samples from cattle (200) and sheep (108) were collected from Shiraz slaughterhouse, southern Iran to investigate the presence of the important Arcobacter spp. using cultivation and Polymerase Chain Reaction (PCR) methods. Antimicrobial susceptibility of Arcobacter isolates was determined for 18 antibiotics using disk diffusion method. Among 308 samples, 27 (8.7%) and 44 (14.28%) were positive for the presence of Arcobacter species with cultivation and PCR procedures, respectively. The predominant species was A. butzleri in both cattle (58.33%) and sheep (55%). In addition, concurrent incidence of the species was observed in 25% of the positive samples. All Arcobacter isolates were resistant to rifampicin, vancomycin, ceftriaxone, trimethoprim and cephalothin. The isolates showed high susceptibility to tetracycline, oxytetracycline, erythromycin, ciprofloxacin, kanamycin, amikacin, gentamicin and enrofloxacin. No significant difference among cattle and sheep isolates in resistance pattern was observed. The results indicate that cattle and sheep are significant intestinal carriers for Arcobacter spp. Moreover, tetracycline and aminoglycosides showed great effects on Arcobacter species in antibiogram test and can be used for treatment of human Arcobacter infections.

Ecological succession leads to chemosynthesis in mats colonizing wood in sea water.

Chemosynthetic mats involved in cycling sulfur compounds are often found in hydrothermal vents, cold seeps and whale falls. However, there are only few records of wood fall mats, even though the presence of hydrogen sulfide at the wood surface should create a perfect niche for sulfide-oxidizing bacteria. Here we report the growth of microbial mats on wood incubated under conditions that simulate the Mediterranean deep-sea temperature and darkness. We used amplicon and metagenomic sequencing combined with fluorescence in situ hybridization to test whether a microbial succession occurs during mat formation and whether the wood fall mats present chemosynthetic features. We show that the wood surface was first colonized by sulfide-oxidizing bacteria belonging to the Arcobacter genus after only 30 days of immersion. Subsequently, the number of sulfate reducers increased and the dominant Arcobacter phylotype changed. The ecological succession was reflected by a change in the metabolic potential of the community from chemolithoheterotrophs to potential chemolithoautotrophs. Our work provides clear evidence for the chemosynthetic nature of wood fall ecosystems and demonstrates the utility to develop experimental incubation in the laboratory to study deep-sea chemosynthetic mats.

Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner.

BACKGROUND: The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain. CONCLUSION/SIGNIFICANCE: Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts.

Short communication: Arcobacter butzleri and Arcobacter cryaerophilus survival and growth in artisanal and industrial ricotta cheese.

Ricotta cheese is a ready-to-eat product with properties (pH >6.0, aw >0.98-0.99) and moisture content (75-80%) that may pose a risk to public health due to postprocess contamination by several bacterial pathogens, including Arcobacters. The objective of the study was to evaluate the behavior of Arcobacter butzleri and Arcobacter cryaerophilus in ricotta cheese during its shelf life assuming postprocessing contamination. Two types of ricotta cheese, artisanal water buffalo (WB) and industrial cow milk ricotta cheese, were experimentally contaminated with A. butzleri and A. cryaerophilus and the count was monitored at 2 different temperatures (6°C and 12°C) during shelf life of 5 d for WB cheese and 22 d for industrial ricotta cheese. In WB ricotta cheese the A. butzleri count remained stable during the 5 d of storage at 6°C, whereas a moderate but significant decrease was observed in A. cryaerophilus count. The counts of both species increased when WB ricotta cheese was stored at 12°C. In industrial ricotta cheese stored at 6°C, a significant reduction was observed both in A. butzleri and A. cryaerophilus counts during the 22-d storage period; at 12°C storage, a count increase was observed for both Arcobacter species up to d 14 of storage after which the log cfu/g count resulted constant until d 22 of storage. The ability of A. butzleri and A. cryaerophilus to survive at 6°C and to grow at 12°C in ricotta cheese has significant food safety implications.

Modified isolation method of Arcobacter spp. from different environmental and food samples.

This study provides information on the occurrence of Arcobacter in several types of water and food products of animal origin in the Czech Republic. We processed 190 samples using the modified method, and the occurrence of Arcobacter spp. was confirmed in 36.8 % of these. This total incidence consisted of Arcobacter butzleri (27.3 %), Arcobacter cryaerophilus (8.4 %) and Arcobacter skirrowii (1.1 %). We newly described the common presence of Arcobacter spp. in sewage water in the Czech Republic that is released into waterways after processing in water treatment plants (86.7 %). All the acquired isolates were subject to detailed confirmation with subsequent species classification using multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we used a modification of a method using passive filtration of an enriched sample, which could be suitable for the isolation of Arcobacter, especially in combination with Campylobacter selective agar chromogenic medium. Our studies have shown this agar to be quite suited to the isolation of Arcobacter and that it can be an appropriate instrument for accelerating culture diagnostics.

Development of an absorbance-based response model for monitoring the growth rates of Arcobacter butzleri as a function of temperature, pH, and NaCl concentration.

In this study, the growth of Arcobacter butzleri in poultry was evaluated as a function of storage temperature (5, 22.5, and 40°C), pH (5, 7, and 9), and NaCl concentration (0, 4, and 8%). A predictive model was developed using the absorbance-based response surface methodology to describe the growth rate. The primary model was obtained to predict a growth rate with a good fit (R2≥0.95), and the secondary model was obtained by nonlinear regression analysis and calculated as follows: Growth rate=-2.267274-0.024181 (Temp)+0.6459384 (pH)+0.1926227 (NaCl)+0.0024661 (Temp×pH)-0.001312 (Temp×NaCl)-0.018802 (pH×NaCl)+0.000467 (Temp2)-0.041711 (pH2)- 0.007426 (NaCl2). Our data showed that the growth of A. butzleri can be completely inhibited at a pH of 5 (in the absence of NaCl, at 5°C) and at a pH of 9 (in the presence of 8% NaCl, at 5°C). The surface response model was statistically significant, with P<0.0001, as evident from the Fisher F test and from coefficient determination (R2, 0.95). This model was also verified by the bias factor (Bf, 0.839), accuracy factor (Af, 1.343), and mean square error (MSE, 0.0138). The newly developed secondary models of growth rate for A. butzleri could possibly be incorporated into a tertiary modeling program such as Pathogen Modeling Program (U.S. Department of Agriculture [USDA]) and Food Micro Model (in the United Kingdom). As a result, they could be used to predict the growth kinetics of A. butzleri as a function of a combination of environmental factors. Ultimately, the developed model can be used to reduce A. butzleri in poultry production, processing, and distribution, thereby enhancing food safety.

Behavior of Arcobacter butzleri and Arcobacter cryaerophilus in ultrahigh-temperature, pasteurized, and raw cow's milk under different temperature conditions.

The growth and survival of Arcobacter butzleri and Arcobacter cryaerophilus in milk were investigated at different storage temperatures. Three strains of each Arcobacter species were inoculated into ultrahigh-temperature (UHT), pasteurized, and raw cow's milk and stored at 4, 10, and 20°C for 6 days. The survival of Arcobacter spp. during storage was evaluated by a culture method. Results clearly showed that A. butzleri and A. cryaerophilus remained viable in milk when stored at 4°C and 10°C for a period of 6 days. When UHT and pasteurized milk were stored at 20°C, the A. butzleri count increased, with a longer lag-phase in pasteurized milk, whereas the A. cryaerophilus count increased in the first 48 h and then rapidly decreased to below the detection limit on the sixth storage day. When raw milk was stored at 20°C, the A. butzleri and A. cryaerophilus counts decreased from the first day of storage and no viable bacteria were recovered on the last day of storage. Generally, A. butzleri displayed a significantly better growth and survival capacity than A. cryaerophilus in milk. The present study is the first to assess the survival and/or growth of A. butzleri and A. cryaerophilus in milk. The evidence suggests that in case of primary contamination of milk or secondary contamination due to postprocessing contamination, milk can act as a potential source of Arcobacter infection in humans and could have public health implications, especially for raw milk consumption.

Higher water temperature and incubation under aerobic and microaerobic conditions increase the recovery and diversity of Arcobacter spp. from shellfish.

Some Arcobacter species are considered emerging food-borne and waterborne pathogens, and shellfish have been suggested as one of their reservoirs. However, only a few studies have investigated the presence of Arcobacter in this kind of food. This study assesses the prevalence and diversity of Arcobacter spp. in shellfish by multiplex PCR (m-PCR) and culturing methods (under different atmospheric conditions) and evaluates the possible influence of environmental parameters (temperature, salinity, and harvesting bay). Arcobacter was detected by m-PCR and/or culturing in 61 (29.9%) of 204 shellfish samples. Of the positive samples by culturing, 41.1% were obtained under only aerobic incubation conditions, while 23.2% were obtained under only microaerobic conditions. Of 476 investigated isolates, 118 belonged to different enterobacterial repetitive intergenic consensus (ERIC)-PCR genotypes (strains) and to 11 different species. This study shows the highest diversity of Arcobacter species ever observed in samples from any origin. The most prevalent species was Arcobacter butzleri (60.2%), followed by Arcobacter molluscorum (21.2%). The prevalence of Arcobacter was significantly higher during the summer than in other seasons, being associated with an increase in water temperature. Results confirm that shellfish are a reservoir for a remarkable diversity of Arcobacter spp.

Bioaccumulation experiments in mussels contaminated with the food-borne pathogen Arcobacter butzleri: preliminary data for risk assessment.

The aim of this study was to evaluate, at a laboratory scale, the ability of this microorganism to grow in seawater and bioaccumulate in mussels (Mytilus galloprovincialis) maintained in constantly aerated tanks, containing twenty litres of artificial seawater. Three concentrations of A. butzleri LMG 10828(T) were tested (about 5 × 106 CFU/mL, 5 × 104 CFU/mL, and 5 × 10² CFU/mL). Following contamination, enumeration of A. butzleri was performed from water and mussels each day, for up to 96 h. Three contamination experiments with artificial seawater in absence of mussels were also performed in the same manner. In the experiments with mussels, A. butzleri declined in water of approximately 1 log every 24 h from the contamination. In artificial seawater without mussels the concentration of A. butzleri remained on the same logarithmic level in the first 48 h and then decreased of about 1 log every 24 hours. In mussels, the concentration was approximately 2 log lower than the exposition level after 24 h from the contamination, and then it decreased exponentially of 1 log every 24 h. Our findings suggest that in the experimental conditions tested A. butzleri is neither able to effectively grow in seawater nor bioaccumulate in mussels, at least in the free and cultivable form.

Survival of Arcobacter butzleri during production and storage of artisan water buffalo mozzarella cheese.

Water buffalo mozzarella cheese (WBMC) is a fresh stretched cheese produced from whole chilled buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. The purpose of this study was to assess the behavior of Arcobacter butzleri during WBMC production and storage under different temperature conditions (5, 10, and 20 °C). Raw milk was experimentally inoculated with one reference strain and two isolates of A. butzleri, and the count was monitored during WBMC production and storage. The bacterial count of A. butzleri decreased during curd ripening (from 7.83 log colony-forming units (CFU)/g to 4.14 log CFU/g in about 4 h) and a further decrease (>4 log CFU/g) was observed at the end of curd stretching. During storage testing, A. butzleri was never detected by direct plating, whereas it was recovered from 12 of the total 162 WBMC until the end of storage testing by enrichment. The results revealed that A. butzleri is able to survive during WBMC production and storage at different temperature conditions. Consequently, traditional WBMC produced from raw milk could represent a potential source of Arcobacter infection for humans.